Power‐effective scanning with AODs for 3D optogenetic applications

Abstract

Two‐photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub‐micrometric‐resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto‐optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD‐based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks.

Acousto-optic deflectors in experimental neuroscience: overview of theory and applications

“…A positive validation of using an AOD system for optical brain stimulation has been reported by direct activation of cell-expressing channelrhodopsins [104,107,108]. In these studies, the ability to selectively activate neurons using a single-photon [104,107] and multiphoton [108] AOD-based system was demonstrated in cultured specimens (cells, neurons, and astrocytes) and the brains of mice, drosophila and zebrafish larvae.…”

“…A positive validation of using an AOD system for optical brain stimulation has been reported by direct activation of cell-expressing channelrhodopsins [104,107,108]. In these studies, the ability to selectively activate neurons using a single-photon [104,107] and multiphoton [108] AOD-based system was demonstrated in cultured specimens (cells, neurons, and astrocytes) and the brains of mice, drosophila and zebrafish larvae. Figure 4(b) shows a HEK 293 cell expressing light-sensitive ChIEF-tdTomato together with the pattern of fixed-spot and whole-soma scanning photostimulation; membrane currents recorded in response to fixed-spot (dark) and whole-soma scanning (red) stimulations at different levels of laser power, together with such currents evoked by traditional Hg lamp illumination (cyan) is also shown.…”

Fast multi-photon brain-wide volumetric imaging and photostimulation

“…This indeed enables the 3D addressing of sequential points in a random-access way (displacing the laser beam at different locations with the same access time, regardless of their relative positions) and in a quasi-simultaneous fashion. Moreover, we devised a novel electronic control for AODs that provides a homogeneous delivery of energy across the volume, thus enabling efficient optogenetic stimulation in 3D using AODs [17]. In fact, AODs usually show a drop in transmission efficiency when moving the beam away from its native focus (along the axial direction).…”

“…Fluorescence signal (reporting calcium activity) is then routed to a sCMOS camera, via an electrically-tunable lens (ETL) which performs remote focusing. The second line (in red) precisely controls the 3D targeting of 1064 nm excitation via two couples of AODs to perform optogenetic stimulation of neurons expressing the red-shifted opsin ReaChR [17]. b.…”

Random-access two-photon holographic optogenetic stimulation combined with brain-wide functional light-sheet imaging in larval zebrafish

“…AODs allows us to randomly access neurons using a cell-shaped hologram at high-speed. 11 This method has already been used to stimulate optogenetically zebrafish larvae, 12 but, to our knowledge functional imaging with a calcium sensor was not recorded at the same time.…”